High-grade malignant gliomas (i.e., astrocytomas) are the most commonly occurring type of lethal adult brain tumor and are increasing in incidence (Legler et al., J. National Cancer Inst., 91:1382-1390 (1999)). The median survival is approximately 9-12 months following diagnosis, as the tumors are usually refractory to aggressive multimodal therapy (Brandes et al., Amer. J. Clin. Onc., 22:387-390 (1999)). Gliomas exhibit increased glycolytic fluxes associated with elevated lactate/pyruvate ratios that would indicate an acidotic intracellular pH (pHi) (Miccoli et al., Biochem. J., 313:957-962 (1996)). However, several 31P spectroscopic (i.e., NMR) studies measured intracellular human glioma pH in situ and reported alkaline values (pH 7.12-7.24) as compared with the surrounding brain (pH 6.99-7.05) (Hubesch et al., Radiology, V174:401-409 (1990); Rutter et al, Invest. Radiology, V30:359-366 (1995)). An alkaline pHi has also been reported in tumor cell lines (Wike-Hooley et al., Radiother. Oncol., 2:343-366 (1984); Vaupel et al., Adv. Exp. Med. Biol., 248:835-845 (1989)). In particular, several human and rat malignant glioma cell lines were reported to exhibit intracellular alkalosis (pHi 7.2-7.5) when compared to rat astrocyte primary cultures (pHi 6.9-7.0) (McLean et al., Amer. J. Physiol. Cell Physiol., 278:C676-688 (2000)).
Malignant gliomas, like many highly proliferative tumors, have increased glycolytic fluxes with elevated levels of intracellular lactate and pyruvate (Oudard et al., Anticancer Res., 17:1903-1911 (1997); Erecinska et al., J. Neurochem., 65:2765-2772 (1995)). Glycolytic rates are optimal at an alkalotic pHi and inhibition of glycolytic flux is sensitive to modest reductions in pHi. (Dobson et al., Amer. J. Physiol., 250:R71-76 (1986)). Obligate tumor DNA synthesis and cell cycle progression are also optimal at an alkalotic pHi (Hasuda et al., One. Res., V6:259-268 (1994)). For example, a reduction in pHi has been associated with reduced rates of proliferation and growth arrest in transformed cell types (Musgrove et al., Exp. Cell Res., 172:65-75 (1987); Rotin et al., Cancer Res., 49:205-211 (1989); Horvat et al., Eur. J. Cancer, 29A: 132-217 (1992)).
The inhibitory effect of a reduction in pHi on tumor cell proliferation is thought to be primarily due to the glycolytic enzyme phosphofructokinase (PFK), which has a pH optimum of 7.2 and is the rate limiting step for glycolysis. In addition, hexokinase activity and intracellular distribution are affected by even modest reductions from an optimal alkaline pHi (Miccoli et al., id), as its activity is required for glucose entry into the glycolytic pathway and is increased in gliomas and in many other proliferative tumors (Katabi et al., Hum. Gene Ther., 10:155-164 (1999); Sebastian et al., Tumour Biol., 19:253-260 (1998)). As such, given the elevated glucose consumption, lactate production, and hypoxic or anoxic environments of malignant gliomas, these tumors may be particularly sensitive to pHi reductions. For example, reducing the pHi in rat C6 gliomas from 7.3 to 6.4 decreased the enzymatic product of PFK by 50% after 15 minutes while doubling the accumulation of substrate (Erecinska et al., id). Lactate and pyruvate levels decreased by 54% and 69%, respectively, during this brief period. Thus, these data confirm that glycolysis in C6 glioma cells is extremely sensitive to modest reductions in pHi.
The alkalosis in glioma cells was reported to result from the persistent activation of NHE1, a ubiquitously-expressed type 1 Na+—H+ exchanger involved in intracellular pH and volume regulation (McLean et al., supra; Hegde et al., J. Pharmacol. Exp. Ther., 310:67-74 (2004)). The Na+—H+ exchanger (NHE) represents a family of sodium-dependent transport proteins that participate in various cellular functions (Orlowski et al., J. Biol. Chem., 272:22373-22376 (1997)). Seven isoforms (i.e., NHE1-7) have been identified (Numata et al., J. Biol. Chem., 276:17387-17394 (2001); Brett et al., Am. J. Physiol., 282:C1031-1041 (2002); Slepkov et al., Biochem. Cell Biol., 80:499-508 (2002)). NHE1 and NHE5-7 are particularly important in maintaining the pHi in human heart and brain. Additionally, increased NHE1 activity has also been observed in other cancer cell lines, including colon and bladder (Bischof et al., Biochimica et Biophysica Acta, 1282:131-139 (1996); Boyer et al., Cancer Res., 52:4441-4447 (1992)).
Amiloride (3,5-diamino-6-chloro-N-(diaminomethylene)pyrazinecarboxamide), originally developed as an antidiuretic drug, displays antiproliferative effects on several cancer cell lines (Horvat et al., id; Hasuda et al., id; Garcá-Cañero et al., Tox. Letters, 106:215-228 (1999); Wong et al., Brit. J. Cancer, 87:238-245 (2002)), including glioma cells (Szolgay-Daniel et al., Cancer Res., 51:1039-1044 (1991)). Amiloride is thought to block tumor cell proliferation through inhibition of specific ion transport systems; in particular, amiloride displays inhibitory activity toward several classes of Na+-dependent membrane transporters, including NHE1, NCX (a Na+-Ca2+ exchanger), the Na+/K+-ATPase, Na+-coupled solute transport, voltage-gated Na+ channels, etc. However, the hydrophobic nature of amiloride, its weak inhibitory activity toward transporters such as NHE1, and its inability to cross the blood brain barrier (BBB) make it unsuitable as an effective drug for treating cancers such as gliomas.
In addition to amiloride, various amiloride derivatives have been synthesized and their activities on ion transporters and glioma cells have been determined. However, such amiloride derivatives are also unsuitable as effective drugs for cancer therapy due to their non-specificity, toxicity, and/or inability to access the central nervous system (i.e., cross the BBB). Agents that selectively inhibit NHE, such as cariporide, do not kill glioma cells and direct acidification does not kill glioma cells (Hegde et al., J. Pharmacol. Exp. Ther., 310:67-74 (2004)). Additional inhibition of NCX is required to confer cytoxicity to cancer cells as is observed with amiloride and dichlorobenzamil, which inhibit both NHE and NCX. Amiloride and dichlorobenzamil are hydrophobic compounds that are rapidly taken up by glioma cells that likely contribute to their nonspecific toxicity (Palandoken et al., J. Pharmacol. Exp. Ther., October 27; Epub. (2004)). Although conjugation of alkyl, alkenyl, or benzyl moieties to either the C(2) guanidine group or the C(5) amino group of amiloride has been reported to increase the inhibitory efficacy of NHE1 and/or other ion transporters (e.g., NCX) (L'Allemain et al., J. Biol. Chem., 259:4313-4319 (1984); Frelin et al., Biochimie, 70:1285-1290 (1988)), these derivatives suffer from the same disadvantages as amiloride (e.g., non-specificity, toxicity, and/or inablity to access the central nervous system). For example, a benyl derivative of amiloride, 2,4-dichlorobenzamil (DCB), is highly toxic and causes lethality when administered.
Thus, there is a need to develop amiloride derivatives (e.g., amiloride conjugates) that (1) target particular cells and/or tissues with high specificity and potency; (2) are low in toxicity to non-targeted cells and/or tissues; (3) are able to be transported across the BBB to access the central nervous system; and (4) kill tumor cell populations residing in hypoxic-ischemic tumor microenvironments that are normally resistant to conventional chemotherapy or radiotherapy. The present invention satisfies this and other needs.